Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields

ABSTRACT

The present invention relates to methods and devices for separating particles according to size. More specifically, the present invention relates to a microfluidic method and device for the separation of particles according to size using an array comprising a network of gaps, wherein the field flux from each gap divides unequally into subsequent gaps. In one embodiment, the array comprises an ordered array of obstacles in a microfluidic channel, in which the obstacle array is asymmetric with respect to the direction of an applied field.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/640,111 filed Dec. 15, 2006, entitled “METHOD FOR CONTINUOUS PARTICLESEPARATION USING OBSTACLE ARRAYS ASYMMETRICALLY ALIGNED TO FIELDS,”which is a continuation of U.S. Pat. No. 7,150,812 filed Oct. 23, 2003,issued Dec. 19, 2006, entitled “METHOD FOR CONTINUOUS PARTICLESEPARATION USING OBSTACLE ARRAYS ASYMMETRICALLY ALIGNED TO FIELDS,”which claims priority to U.S. Provisional Patent Application No.60/420,756 filed Oct. 23, 2002, the entire disclosures of which arehereby incorporated by reference, for all purposes, as if fully setforth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant#MDA972-00-1-0031 awarded by the Defense Advanced Research ProjectsAgency (DARPA). The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods and devices for separatingparticles according to size. More specifically, the present inventionrelates to a microfluidic method and device for the separation ofparticles according to size.

BACKGROUND OF THE INVENTION

Separation by size or mass is a fundamental analytical and preparativetechnique in biology, medicine, chemistry, and industry. Conventionalmethods include gel electrophoresis, field-flow fractionation,sedimentation and size exclusion chromatography [J. C. Giddings, UnifiedSeparation Science (Wiley, New York, 1991)]. Gel electrophoresisutilizes an electric field to drive charged molecules to be separatedthrough a gel medium, which serves as a sieving matrix. The moleculesare initially loaded at one end of a gel matrix, and are separated intocomponent zones as they migrate through the gel. Field-flowfractionation is carried out in a thin ribbon-like channel, in which theflow profile is parabolic. Particles are loaded as a sample zone, andthen flow through the channel. Separation occurs as particles ofdifferent properties flow in different positions of the flow, due to theinfluence of a field, resulting in different migration speeds. The fieldis applied perpendicular to the flow. Sedimentation utilizesgravitational or centrifugal acceleration to force particles through afluid. Particles migrate through the fluid at different speeds,depending on their sizes and densities, and thus are separated. Sizeexclusion chromatography (SEC) utilizes a tube packed with porous beads,through which sample molecules are washed. Molecules smaller than thepores can enter the beads, which lengthen their migration path, whereasthose larger than the pores can only flow between the beads. In this waysmaller molecules are on average retained longer and thus becomeseparated from larger molecules. Zones broaden, however, as they passthrough the column, because there are many possible migration paths foreach molecule and each path has a different length, and consequently adifferent retention time. This multipath zone broadening (Eddydiffusion) is a major factor limiting resolution. J. C. Giddings,Unified Separation Science (John Wiley & Sons, New York, 1991). Othermethods for separation according to size, including gel electrophoresis,field-flow fractionation, also involve stochastic processes, which maylimit their resolution. J. C. Giddings, Nature 184, 357 (1959); J. C.Giddings, Science 260, 1456 (1993).

The need for reliable and fast separation of large biomolecules such asDNA and proteins cannot be overemphasized. Recently,micro/nano-fabricated structures exploiting various ideas for DNAseparation have been demonstrated. The use of micro/nano-fabricatedstructures as sieving matrices for particle separation was disclosed inU.S. Pat. No. 5,427,663. According to this document, DNA molecules areseparated as they are driven by electric fields through an array ofposts. U.S. Pat. No. 5,427,663 discloses a sorting apparatus and methodfor fractionating and simultaneously viewing individual microstructuresand macromolecules, including nucleic acids and proteins. According toU.S. Pat. No. 5,427,663, a substrate having a shallow receptacle locatedon a side thereof is provided, and an array of obstacles outstandingfrom the floor of the receptacles is provided to interact with themicrostructures and retard the migration thereof. To create migration ofthe microstructures, electrodes for generating electric fields in thefluid are made on two sides of the receptacle. This is analogous to theconventional gel electrophoresis. However, micromachined structures aresubstituted for gel as sieving matrices.

A variety of microfabricated sieving matrices have been disclosed. Inone design, arrays of obstacles sort DNA molecules according to theirdiffusion coefficients using an applied electric field [Chou, C. F. et.al., Proc. Natl. Acad. Sci. 96, 13762 (1999).]. The electric fieldpropels the molecules directly through the gaps between obstacles,wherein each gap is directly below another gap. The obstacles are shapedso that diffusion is biased in one direction as DNA flows through thearray. After flowing through many rows of obstacles, DNA with differentdiffusion coefficients are deflected to different positions. However,because the diffusion coefficient is low for large molecules, theasymmetric obstacle arrays are slow, with running times of typicallymore than 2 hours. In a second design, entropic traps consisting of aseries of many narrow constrictions (<100 nm) separated by wider anddeeper regions (a few microns), reduce the separation time to about 30minutes [Han, J. & Craighead, H. G., Science 288, 1026 (2000).]. Becausethe constrictions are fabricated to be narrower than the radius ofgyration of DNA molecules to be separated, they act as entropicbarriers. The probability of a molecule overcoming the entropic barrieris dependent on molecular weight, and thus DNA molecules migrate in theentropic trap array with different mobilities. Larger molecules, withmore degrees of configurational freedom, migrate faster in thesedevices. In a third design, a hexagonal array of posts acts as thesieving matrix in pulsed-field electrophoresis for separation of DNAmolecules in the 100 kb range [Huang, L. R., Tegenfeldt, J. O., Kraeft,J. J., Sturm, J. C., Austin, R. H. and Cox, E. C., Nat Biotechnol. 20,1048 (2002).]. However, these devices generally require features sizescomparable to or smaller than the molecules being fractionated. Han, J.& Craighead, H. G. Separation of long DNA molecules in a microfabricatedentropic trap array. Science 288, 1026-1029 (2000); Turner, S. W.,Cabodi, M., Craighead, H. G. Confinement-induced entropic recoil ofsingle DNA molecules in a nanofluidic structure. Phys Rev Lett. 2002Mar. 25; 88(12):128103; Huang, L. R., Tegenfeldt, J. O., Kraeft, J. J.,Sturm, J. C., Austin, R. H. and Cox, E. C. A DNA prism for high-speedcontinuous fractionation of large DNA molecules. Nat Biotechnol. 2002October; 20(10):1048-51; and Huang, L. R., Silberzan, P., Tegenfeldt, J.O., Cox, E. C., Sturm, J. C., Austin, R. H. and Craighead, H. Role ofmolecular size in ratchet fractionation. Phys. Rev. Lett. 89, 178301(2002). The need for small feature size may have the followingdetrimental effects: (i) the devices cannot fractionate small moleculessuch as proteins, (ii) the devices may have very low throughput, andthus are not useful sample preparation tools, (iii) the devices can onlyanalyze very small volume of samples, and therefore usually requireconcentrated samples or expensive equipment for sample detection, and(iv) manufacturing the devices require state-of-the-art fabricationtechniques, and thus high cost.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a microfluidic device for separatingparticles according to size comprising a microfluidic channel, and anarray comprising a network of gaps within the microfluidic channel. Thedevice employs a field that propels the particles being separatedthrough the microfluidic channel. The individual field flux exiting agap is divided unequally into a major flux component and a minor fluxcomponent into subsequent gaps in the array, such that the averagedirection of the major flux components is not parallel to the averagedirection of the field.

In a preferred embodiment, the present invention provides a microfluidicdevice for separating particles according to size comprising amicrofluidic channel, and an ordered array of obstacles within themicrofluidic channel. The ordered array of obstacles is asymmetric withrespect to the average direction of the applied field. The deviceemploys a field that propels the particles being separated through themicrofluidic channel.

The present invention also provides a method for separating particlescomprising introducing the particles to be separated into an arraycomprising a network of gaps within the microfluidic channel andapplying a field to the particles to propel the particles through thearray. A field flux from the gaps is divided unequally into a major fluxcomponent and a minor flux component into subsequent gaps in the array,such that the average direction of the major flux components is notparallel to the average direction of the field.

In a preferred embodiment, the present invention also provides a methodfor separating particles according to size comprising: introducing theparticles to be separated into a microfluidic channel comprising anordered array of obstacles, and applying a field to the particles,wherein the ordered array of obstacles is asymmetric with respect to theaverage direction of the applied field.

In another embodiment, the present invention provides a microfluidicdevice for separating particles according to size comprising amicrofluidic channel, and multiple arrays in series within themicrofluidic channel, wherein each array has a different critical size.The device employs a field that propels the particles being separatedthrough the microfluidic channel. Each of the arrays comprises a networkof gaps wherein a flux of the field from the gaps is divided unequallyinto a major flux component and a minor flux component into subsequentgaps in the network. The average direction of the major flux componentsin each array is not parallel to the average direction of the field.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described in conjunction with the appendedfigures:

FIG. 1 shows a schematic diagram of an array device according to anembodiment of the present invention. The device consists of an obstaclearray asymmetric about the field direction.

FIG. 2 shows a schematic diagram of a perspective view of a preferredembodiment of the invention, comprising a base substrate with a mainsurface, on which an obstacle course is made to form a network of gaps,wherein cascades of unequal bifurcations of field flux occur. Theobstacle course may be sealed to a cap layer to form an enclosed networkof gaps.

FIG. 3 shows a schematic diagram of an obstacle array. The obstaclearray is symmetric about the dotted lines (principle axis), butasymmetric with respect to the field direction.

FIG. 4 shows a schematic diagram of an embodiment of the invention,comprising a network of gaps defined by an array of obstacles. Eachstreamline represents an equal amount of field flux. The field flux fromone gap is divided into two subsequent gaps, wherein the amounts of fluxgoing into the two gaps are unequal. In this case, two streamlines goesto the left (major flux component) and one streamline goes to the right(minor flux component) at each gap. The array direction is indicated bythe gray arrow.

FIG. 5 shows a schematic diagram of particles separated in an arraydevice according to an embodiment of the present invention. The smallparticles move along field and large ones towards array direction.

FIG. 6 shows a schematic diagram of an obstacle array, showingcharacteristic array dimensions. .lamda. denotes the period of a row ofobstacles, d the gap spacing between obstacles, and a.lamda. the lateralshift of every row.

FIG. 7 shows a schematic diagram of a particular array, showing thatfield lines go around obstacles. The dotted line at the center marks thedivision of field lines going to different sides of the obstacle.

FIG. 8 shows a schematic diagram of a particular array illustrating thatfield lines shift relative positions in gaps. To illustrate, a=⅓ andeach gap is divided into three slots, which are denoted 1, 2 and 3.Field lines passing through slot 1 go to slot 2 in the next gap, thosethrough slot 2 go to 3, and the ones through 3 to 1.

FIG. 9 shows a schematic diagram of a particular array illustrating thepath of small particles.

FIG. 10 shows a schematic diagram of a particular array illustrating thedisplacement of large particles by obstacles.

FIG. 11 shows a schematic diagram of a particular array illustrating thepath of large particles. Large particles move along the array direction(displacement mode). Note that even if the particle starts at the leftside of the gap, eventually it moves to the right side and stays at theright as it is constantly being displaced by obstacles.

FIG. 12 shows a plot of particle migration direction vs. their size.There exists a critical particle size R.sub.0 where a sharp transitionof migration direction occurs.

FIG. 13 shows a schematic diagram of two arrays of different criticalsizes in series. Particles at output 1 are smaller than R.sub.1, thecritical size of the first array in the series, those at output 2between size R.sub.1 and R.sub.2, and the ones at output 3 are largerthan R.sub.2, the critical size of the second array.

FIG. 14 shows a schematic diagram of multiple arrays in series, whereineach array has a different critical size. The dotted horizontal linerepresents the division between subsequent arrays and each subsequentarray in the series has an increasing critical size. A continuousdistribution of molecular sizes can thus be fractionated and analyzedwith one device.

FIG. 15 shows a schematic top view of an embodiment of the invention,using a square array of circular obstacles to form the network of gaps,tilted at a small angle .theta. with respect to the field. The squarearray is spanned by primitive vectors x and y, which are perpendicularto each other. Bifurcation of field flux occurs because of the asymmetryof the obstacle array with respect to the field.

FIG. 16 shows a schematic top view of an embodiment of the invention.Each obstacle is centered at a lattice point, spanned by the primitivevectors x and y. However, the obstacles may have different shapes.

FIG. 17 shows a schematic top view of an embodiment of the invention.Every obstacle may be identical with the same horizontal period .lamda..However, every row of obstacles is shifted horizontally by a differentamount with respect to the previous row, and the obstacle course is nota periodic lattice.

FIG. 18 shows a schematic diagram of a microfluidic device forcontinuous-flow separation, with sample loading structures.

FIG. 19 shows a diagram of a microfluidic device for continuous-flowseparation as constructed in Example 1.

FIG. 20 shows fluorescent images of microspheres migrating in theobstacle array, showing (A) the two transport modes for (B) theseparation of microspheres with no dispersion. The gray dots in (A),which represent the obstacles, have been superimposed on the fluorescentimage.

FIG. 21 shows the fluorescent profiles of microspheres separated usingflow speeds of .about.40 .mu.m/s (upper curves) and .about.400 .mu.m/s(lower curves) scanned at .about.11 mm from the injection point. The0.60 .mu.m, 0.80 .mu.m, and 1.03 .mu.m diameter beads aregreen-fluorescent, while 0.70 .mu.m and 0.90 .mu.m are red, and thuseach scan is shown as two curves representing the two colors.

FIG. 22 shows the measured migration angles as a function of microspherediameter at two different flow speeds.

FIG. 23 shows a schematic diagram of a device for particle separationcomprising an array having 9 sections (divided by dotted lines) ofdifferent critical sizes, and sample loading structures. While theorientation and the lattice constants of the array are kept the same,the obstacle diameters are changed to create different-sized gaps d andcritical sizes.

FIG. 24 shows the high-resolution separation of fluorescent microspheresof 0.80 .mu.m (left), 0.90 .mu.m (center) and 1.03 .mu.m (right), usingan array of varying gap size. While the orientation and the latticeconstants of the array are kept the same, the obstacle diameters arechanged to create different-sized gaps d, labeled on the left side ofthe fluorescent image. Individual 1.03 .mu.m streamlines clearly showzigzag migration.

FIG. 25 shows the fluorescent profile of the separated particles scannedat 14 mm from the injection point.

FIG. 26 shows of fluorescent image of DNA molecules separated accordingto size using a device of the present invention. Molecules areintroduced at the top of the figure.

FIG. 27 shows a schematic diagram of a device used to concentratesample. The field direction can be defined by the sidewalls of thearray. The shaded area shows the trajectories of particles larger thanthe critical size, injected from the top. Particles move against thesidewall and are concentrated.

FIG. 28 shows a schematic diagram of a device for concentratingparticles. The device comprises an array, which moves particles againsta boundary of the array, and microfluidic channels and reservoirs forsample loading and collection.

FIG. 29 shows of fluorescent image of DNA molecules getting concentratedagainst a boundary of the array. DNA samples were introduced from thetop of the picture.

FIG. 30 shows a schematic diagram of a device having an ordered array ofobstacles and employing a non-uniform field, wherein the direction ofthe field changes across the array.

FIG. 31 shows a schematic diagram of a device having an ordered array ofobstacles in a curved microfluidic channel. The curved channel mayresult in a field that is non-uniform.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method and a device for separatingparticles according to size using an array comprising a network of gaps,wherein the field flux from a gap divides unequally into subsequentgaps. A field is applied to the array to propel the particles beingseparated through the array. In a preferred embodiment, the array is anordered array of obstacles in a channel, wherein the array is asymmetricwith respect to the direction of the applied field. In further preferredembodiments, the channel that contains the array is a microfluidicchannel. The term “channel” as used herein refers to a structure whereinfluid may flow. A channel may be a capillary, a conduit, a strip ofhydrophilic pattern on an otherwise hydrophobic surface wherein aqueousfluids are confined, etc. The term “microfluidic” as used herein, refersto a system or device having one or more fluidic channels, conduits orchambers that are generally fabricated at the millimeter to nanometerscale, e.g., typically having at least one cross-sectional dimension inthe range of from about 10 nm to about 1 mm.

The present invention is useful for the separation of biologicalparticles according to size, including bacteria, cells, organelles,viruses, nucleic acids (i.e., DNA, etc.), proteins and proteincomplexes, as well as other nonbiological particles suspended in fluid,such as industrial polymers, powders, latexes, emulsions, and colloids.In addition to particle separation, the method can be used to analyzethe size distribution of samples, or extract particles of certain sizerange from mixtures of particles. Further, because of the large featuressize, the device may be a high throughput sample preparation tool. Thepresent invention may provide the advantages of low manufacturing cost,high resolution, and improved sample throughput.

In a preferred embodiment, the present invention provides a microfluidicdevice that separates particles in fluid according to size (for example,see FIG. 1). A field is applied to the particles being separated as theparticles pass through the array. The term “field” as used herein refersto any force or vector entity that can be used to propel the particlesthrough the array. The field that drives the particles being separatedmay be a force field, such as an electric field, a centrifugal field, ora gravitational field. The field that drives the particles beingseparated may also be a fluid flow, such as a pressure-driven fluidflow, an electro-osmotic flow, capillary action, etc., wherein thevector entity is the fluid flux density. Further, the field may be acombination of a force field and a fluid flow, such as anelectro-kinetic flow, which is a combination of the electric field andthe electro-osmotic flow. In preferred embodiments, the averagedirection of the field will be parallel to the walls of the channel thatcontains the array.

The array for use in the present invention comprises a network of gapsthat creates a field pattern such that the field flux from a gap withinthe network is divided into unequal amounts (a major flux component anda minor flux component) into the subsequent gaps (for example, see FIG.4), even though the gaps may be identical in dimensions. Looking at thearray as a whole, on average the unequal divisions of the field flux isweighted in one direction. Thus, the major flux components are diverted,on average, in the same direction (i.e., diverted to the same side ofthe obstacle), such that the average direction of the major fluxcomponents is not parallel to the average direction of the minor fluxcomponents, or to the average direction of the field. It is preferredthat a majority of the major flux components be diverted in the samedirection. In a particularly preferred embodiment, the array is anordered array, wherein the major flux component from each bifurcationevent is diverted in the same direction. Generally, the minor fluxcomponent exiting one gap feeds into the major flux component exiting asubsequent gap (FIG. 4).

The term “gap” as used herein refers to a structure wherein fluids orparticles may flow. A gap may be a capillary, a space between twoobstacles wherein fluids may flow, a hydrophilic pattern on an otherwisehydrophobic surface wherein aqueous fluids are confined, etc. In apreferred embodiment of the invention, the network of gaps is defined byan array of obstacles. In this embodiment, the gaps would be the spacebetween adjacent obstacles. In a preferred embodiment, the network ofgaps will be constructed with an array of obstacles on surface of asubstrate (FIG. 2). The term “obstacle” as used herein refers to aphysical structure or pattern wherein the particles being separatedcannot penetrate, and thus an obstacle may refer to a post outstandingon a base substrate, a hydrophobic barrier for aqueous fluids, etc. Insome embodiments, the obstacle may be permeable to the field. Forexample, an obstacle may be a post made of porous material, wherein thepores allows penetration of the field, but are too small for theparticles being separated to enter.

In a preferred embodiment, the network of gaps, wherein the field fluxis divided unequally, is formed using a periodic array of obstacles,which is asymmetric with respect to the average direction of the field.An important feature of this embodiment is that the obstacle array isasymmetric with respect to the field, even though the array itself maybe symmetric with respect to other axes (FIG. 3). The term asymmetric asused herein refers to an obstacle array in which the field flux from agap between two obstacles is bifurcated by a subsequent obstacle, suchthat the bifurcated field flux is not divided into two equal amounts(for example, see the field stream lines of FIG. 4). This may beachieved when the average direction of the field is not parallel to aprinciple axis of the array (FIG. 3). In one embodiment, an orderedarray is tilted at an offset angle .theta. with respect to the field(for example, FIG. 15), wherein the offset angle .theta. is selectedsuch that the array is not aligned to the field (i.e., is asymmetric tothe field). Alternatively, an asymmetric array may also be achievedusing an array comprising rows of obstacles in which each row islaterally shifted from the previous row and the misalignment factor, a,is larger than 0 and smaller than 0.5 (FIG. 6). In a preferredembodiment, the array of obstacles will be an ordered array in which aprinciple axis of the array is not parallel to the direction of thefield (FIG. 3). The term asymmetric refers to the array of obstacles(for example, to the array axis) and not to the shape of individualobstacles. It is preferred that the array is an ordered array in orderto maximize the number of bifurcation events that can occur as thesample passes through the array (for example, see FIG. 4).

As used herein, the term “ordered” refers to an array having a generallyperiodic or repeating spatial arrangement. For example the repeatingspatial arrangement, may be square, rectangular, hexagonal, oblique,etc. In other embodiments, the array need not be an ordered array.

In one embodiment of the invention, particles flow through an asymmetricobstacle array, and are separated according to size into differentstreams (FIG. 5). While small particles follow the field direction,large particles migrate at the array direction. The array directioncorresponds to the average direction of the major component of the fieldflux (for example, see the gray arrow of FIG. 4). The basic theory ofthe transport process is schematically illustrated in FIG. 5. FIG. 5depicts one embodiment of an asymmetric array of obstacles in which thearray is misaligned (i.e., asymmetric) to the field. For the array typedepicted in FIG. 6, the misalignment factor, a, is larger than 0 andsmaller than 0.5. When a=0 or a=0.5, the array is symmetric about thefield axis. As shown in FIG. 6, each row of obstacles is shiftedhorizontally with respect to the previous row by a.lamda., where .lamda.is the center-to-center distance between the obstacles. For the purposeof this discussion, let us assume that a equals ⅓. In preferredembodiments, particles to be separated are driven through the array by afluid flow. Because of the low Reynolds number in these devices, theflow lines may be laminar, resulting in negligible turbulence andinertial effects.

Because the obstacle array is asymmetrically aligned to the field, i.e.,the obstacle lattice is asymmetric with respect to the average fielddirection, field lines going through one gap have to go around theobstacle in the next row (FIG. 7). To consider the allocation of fieldlines in the next row, we assume that the field in the gap betweenobstacles is locally uniform, i.e. field lines are equally spaced inevery gap. This may be a good approximation if the field is electrical.This assumption may be relaxed in the case of other fields. The totalfield flux .PHI. going through one gap is divided by the subsequentobstacle and splits into two streams, the major flux component and theminor flux component, as the flux goes into the two subsequent gaps(FIG. 7). If that the average field direction is vertical (parallel tothe channel walls), a.PHI. has to go to one gap and (1−a).PHI. to theother. Therefore, unequal division of the field flux occurs at each gap.Generally, the minor flux component exiting one gap feeds into the majorflux component exiting a subsequent gap (FIG. 4).

If we divide each gap into 1/a slots (FIG. 8, to illustrate a=⅓), eachfield line shifts to the next slot as it passes through a row in acyclic manner: field lines going through position 1 will be at position2 in the next row, those at position 2 will go to position 3 . . . theones at position 1/a will go back to position 1. Small particles followthe field and shift from one position to the next in a cyclic manner(FIG. 9). Because small particles may go back to their original slot(relative position in the gap) after 1/a rows, their trajectories followthe field direction and do not disperse after going around manyobstacles. Because of the “zigzag” motion of the small particles, thistransport pattern may be referred to as the “zigzag mode.”

In contrast, particles with a large diameter compared to the slot widthwill not follow individual streamlines, but instead be propelled by manystreamlines, which fundamentally changes their final direction ofmigration. As shown in FIG. 10, a particle in contact with an obstacleand whose characteristic radius R is larger than ad (the width of thegap), will follow a field line going through slot 2, because its centerfalls in slot 2. Now the field lines goes to slot 3 in the next gap, sothe center of the particle should also be in slot 3. However, slot 3does not have enough room for the particle. Therefore the particle isdisplaced back to slot 2. This process may be repeated every time as alarge particle approaches a row of obstacles. Because large particlesare displaced from slot 3 to slot 2, they follow the array direction(FIG. 11). The motion of large particles through the array is termed the“displacement” mode.

In summary, there exists a critical particle radius R.sub.0 above whichparticles move in the array direction (displacement mode), and smallerthan which particles follow the average field direction (zigzag mode)(FIG. 12). The critical size, R.sub.0, may be determined by a and d,where a is the misalignment factor and d is the gap width. In the casewhere fields are evenly distributed in gaps, R.sub.0=ad. Thus, aparticle's hydrodynamic radius (size) determines which transport mode itfollows. Also note that the above theory can be easily generalized forother fields, such as electrophoretic fields or pressure driven fluidflows by considering ion flows instead of fluid flows.

In one embodiment of the invention, the device may be employed as afilter. The term “filter” as used herein refers to a device whichremoves particles in certain size ranges from a fluid. The sharptransition of migration angle with respect to size is ideal for filters(FIG. 12). The micro/nano-structures of the arrays are preferably madeby micro/nano-fabrication techniques. FIG. 13 shows the schematicdiagram of a filter of the invention, wherein injected particles areseparated into three groups of different sizes. In principle, one arraycan set up one critical radius (size) for particle separation, andseparate a mixture of particles into two groups of particles, in one ofwhich particles are larger than the critical size, and in the other ofwhich particles are smaller. Two arrays of different critical sizes puttogether in series can separate a mixture of particles into three sizeranges, large, medium, and small (FIG. 13). The sample of particles ofdifferent sizes is injected in the first array, which has a smallercritical size (R.sub.1) than the second (R.sub.2). While the large andmedium-sized particles move in the displacement mode in the first array,the small ones move in the zigzag mode and are separated out. Asparticles move into the second array, the medium-sized particles switchto the zigzag mode and are separated out from the large ones. One cancollect molecules of different sizes at the output of the second array,which may serve as a molecular filter and a sample preparation tool.Further, the size range of particles going to output 2, determined byR.sub.1 and R.sub.2, can be very narrow. For example, the device can bedesigned to single out only one size of DNA restriction fragments fromtens or hundreds of other sizes of fragments. This device could replacethe conventional techniques of gel electrophoresis and gel cutting.

In another embodiment of the invention, the device of the invention mayfractionate molecules according to size. High selectivity can beachieved at the sharp transition region (FIG. 12), using a single arrayof fixed critical radius (size). Alternatively, separation of particlesin a broad size-range may require lower selectivity, which could beachieved using many arrays in series, each of which has a differentcritical size (FIG. 14). FIG. 14 shows a schematic diagram of multiplearrays in series, wherein each array has a different critical size. Thedotted horizontal line represents the division between subsequent arraysand each subsequent array in the series has a increasing critical size.A continuous distribution of molecular sizes can thus be fractionatedand analyzed with one device. In fact, smoothmigration-angle-to-particle-size characteristics, including linear andexponential relationships, can be designed using many arrays in series,each having a different critical size. The critical size of a array maybe adjusted by changing the gap width d, by shifting obstacles, or both.

In one embodiment of the invention, the device may comprise an orderedarray of obstacles and may employ a non-uniform field, wherein thedirection of the field changes across the array (FIG. 30). In oneembodiment, the changing field directions may be created by a curvedmicrofluidic channel. Since the critical size of the array depends onthe field direction, the non-uniform field creates sections of array ofdifferent critical sizes. This may be desired for fractionation ofparticles in a broad size range. In the particular embodiment shown inFIG. 30, the field direction is large at the first array section andthen reduced at the following array sections, creating a large criticalsize in the first section and smaller critical sizes at the followingsections.

In another embodiment of the invention, the device comprises an orderedsquare array of obstacles in a curved microfluidic channel (FIG. 31).The curved channel may result in a field that is non-uniform. The changein the field direction across the array creates a gradient of criticalsize in the array, because the critical size may depend on the fielddirection. Further, the field strength, another parameter that can betuned, may be adjusted by changing the width of the microfluidicchannel. This device may have better separation range and resolutionthan an array of one fixed critical size. A continuous gradient ofcritical size across the array can also be created by changing gapwidths.

In one embodiment of the invention, the array comprises a square latticeof cylindrical obstacles, with the array tilted at an offset angle.theta. with respect to the field (FIG. 15). An offset angle .theta. oftan.sup.−1 (a) is equivalent to a misalignment factor of a in FIG. 6.

In another embodiment of the invention, the array comprising a networkof gaps may be formed by a course of obstacles, which are not identical(FIG. 16). More specifically, obstacles may have different shapes ordimensions. Because the lattice on which obstacles overlay is asymmetricwith respect to the field, the field flux in each gap between obstaclesis divided unequally into the subsequent gaps.

In another embodiment of the invention, the obstacle course, which formsthe network of gaps, may not be a periodic lattice. For example, FIG. 17shows a schematic top view of an embodiment of the invention in whicheach obstacle may be identical and have the same horizontal period.lamda.. However, every row of obstacles is shifted horizontally by adifferent amount with respect to the previous row, and the obstaclecourse is not a Bravais lattice [N. W. Ashcroft and N. D. Mermin, SolidState Physics (Saunders College Publishing, 1976)]. Nonetheless, fieldflux in the array undergoes cascades of bifurcations because theobstacle course is asymmetric with respect to the average fielddirection. Because the critical size is dependent on the asymmetry ofthe field flux division, which originated from the asymmetry of thearray with respect to the field, this embodiment may be useful forseparating particles in a broad range.

In another embodiment of the invention, particles are loaded into thearray using many microfluidic channels at the top of the array (FIG.18). The many channels generate a field pattern that is generallyuniform across the array. Particles are injected from one or morechannels connected to one or more reservoirs containing the particles,which are to be carried across the array by the field.

In another embodiment of the invention, particles are unloaded from thearray, for example via microfluidic channels at the end of the array(FIG. 18). The particles can then be routed to the next component of themicrofluidic chip for further use.

In another embodiment, the device of the invention can concentrateparticles larger than a critical size (FIG. 27). This embodiment mayexploit the array's ability to move particles off the applied fielddirection in displacement mode. Particles are introduced on one side ofthe array. Particles larger than the critical size of the array (movingin displacement mode) get piled against another boundary of the array.The device of the invention in this case acts as a concentrator. Theconcentrated sample can then be routed to the next component of themicrofluidic chip for further use, such as creating a sample zone forfractionation. The concentrated sample can also be taken off the chipfor further applications.

In a preferred embodiment of the invention, the device ismicro/nano-fabricated. Microfabrication techniques may be selected fromthose known in the art, for example, techniques conventionally used forsilicon-based integrated circuit fabrication, embossing, casting,injection molding, and so on [E. W. Becker et. al., MicroelectronicEngineering 4 (1986), pages 35 to 56]. Examples of suitable fabricationtechniques include photolithography, electron beam lithography, imprintlithography, reactive ion etching, wet etch, laser ablation, embossing,casting, injection molding, and other techniques [H. Becker et. al., J.Micromech. Microeng. 8 (1998), pages 24 to 28]. The microfluidic devicemay be fabricated from materials that are compatible with the conditionspresent in the particular application of interest. Such conditionsinclude, but are not limited to, pH, temperature, application of organicsolvents, ionic strength, pressure, application of electric fields,surface charge, sticking properties, surface treatment, surfacefunctionalization, and bio-compatibility. The materials of the deviceare also chosen for their optical properties, mechanical properties, andfor their inertness to components of the application to be carried outin the device. Such materials include, but are not limited to, glass,fused silica, silicone rubber, silicon, ceramics, and polymericsubstrates, e.g., plastics, depending on the intended application.

It is understood that the various embodiments described herein are byway of example only, and are not intended to limit the scope of theinvention. For example, many of the materials and structures describedherein may be substituted with other materials and structures withoutdeviating from the spirit of the invention. It is understood thatvarious theories as to why the invention works are not intended to belimiting.

EXAMPLES

Specific representative embodiments of the invention will now bedescribed, including how such embodiments may be made. It is understoodthat the specific methods, materials, conditions, process parameters,apparatus, and the like do not necessarily limit the scope of theinvention.

Example 1

A microfluidic device was constructed (FIG. 19). The microfluidicchannel is 16 mm long, 3.2 mm wide, and 10 .mu.m deep. The array fillingthe channel consists of a square lattice of cylindrical obstacles (FIG.19, sub-view A), where the center-to-center distance, .lamda. is 8.mu.m, and the spacing d between the obstacles 1.6 .mu.m (FIG. 19,sub-view B). The field employed here is a pressure driven field flow.The lattice is rotated by 5.7.degree. (tan.sup.−1 0.1) with respect tothe channel (FIG. 19, sub-view A), which defines the flow direction. Therotation of tan.sup.−1 0.1 corresponds to a=0.1 thus a complete pitch.lamda. is shifted every 10 rows (FIG. 19, sub-view A). In principle,this configuration provides 10 slots, rather than the 3 discussed above.Particles are injected from a 10 .mu.m-wide channel and carried acrossthe array by a pressure-driven flow, which is made uniform by the manychannels on the top and bottom of the array (FIG. 19, sub-view C). Themicrofluidic channels and the array were fabricated on a silicon waferusing photolithography and deep reactive ion etching, techniquesconventionally used for silicon-based integrated circuit fabrication.Holes through wafers for fluid access were drilled before sealing with aglass coverslip coated with silicone rubber (RTV-615 from GeneralElectric) to sandwich the array.

The two transport modes were experimentally observed using fluorescentpolystyrene microspheres of 0.40 .mu.m and 1.03 .mu.m in aqueous buffer(FIG. 20, sub-view A) (0.1.times. ris-Borate-EDTA buffer containing0.02% POP-6, a performance-optimized linear polyacrylamide (Perkin-ElmerBiosystems), was used in the experiments.). The image was taken byfluorescent microscopy with a long exposure time to show trajectories ofindividual particles. The varying brightness along the trajectoryreflects the different flow speeds of a microsphere in the array, dimmerin the narrow gaps due to higher flow speed and lower residence time. Aspredicted, the 0.40 .mu.m microsphere (left) crossed a column ofobstacles every 10 rows in the zigzag mode, whereas the 1.03 .mu.mmicrosphere (right) was channeled along the axis of the obstacle arrayin the displacement mode.

FIG. 20, sub-view B shows the continuous-flow separation of 0.40 .mu.mand 1.03 .mu.m microspheres injected into the array from a feed channelat the top. In this image many trajectories were superimposed. The 0.40.mu.m and 1.03 .mu.m spheres migrated at .about.0.degree. (flowdirection) and .about.5.7.degree. (array rotation) respectively relativeto the flow direction, as expected. The pressure used to drive the flowwas 30 kPa, which created an average flow speed of .about.400 .mu.m/s.The running time through the device was .about.40 s.

To probe the resolution of the device, fluorescent microspheres of 0.60.mu.m, 0.70 .mu.m, 0.80 .mu.m, 0.90 .mu.m and 1.03 .mu.m diameter weremixed and injected into the array (The concentrations of themicrospheres of 0.60 .mu.m, 0.70 .mu.m, 0.80 .mu.m, 0.90 .mu.m and 1.03.mu.m were 0.015%, 0.010%, 0.010%, 0.005%, and 0.005% solid,respectively). The beads were separated into different streams using aflow speed of .about.40 .mu.m/s, created by a driving pressure of 3 kPa.The fluorescence profile scanned 11 mm from the injection point is shownin FIG. 21. The measured migration directions with respect to the flow(field), defined as the migration angles, are plotted as a function ofthe microsphere size in FIG. 22, which shows that under this flow speed(.about.40 .mu.m/s), the transition from the zigzag to the displacementmode is gradual. The smooth transition is probably due to Brownianmotion of the particles between streamlines. At a flow speed of 40.mu.m/s, a 0.6 .mu.m particle—diffusion coefficient in water of 0.73.mu.m.sup.2/s (H. C. Berg, Random Walks in Biology, Princeton UniversityPress, New Jersey, 1993, p. 56)—has a diffusion length of .about.0.54.mu.m over the 0.2 s it takes to move the 8 .mu.m from one row ofobstacles to the next. This is a factor of 3 larger than the averageslot width of .about.0.16 .mu.m.

To minimize the effects of Brownian motion, the Peclet number wasincreased by increasing the flow speed. Peclet number (Pe) is defined asPe=vd D, where v is the flow speed, d is the characteristic dimension ofthe array, and D the diffusion coefficient of the particle beingseparated. FIG. 22 shows a sharper transition when the flow speed isincreased by a factor of 10 (.about.400 .mu.m/s), created by a drivingpressure of 30 kPa. The high flow speed not only increases theselectivity so that the device becomes more sensitive to size changes,but also shortens the running time to .about.40 s. The transition occursapproximately at 0.8 .mu.m (FIG. 22). The size coefficients of variation(CV) of the best monodispersed microspheres commercially available are1.3%, 1.0% and 1.0% for 0.70 .mu.m, 0.80 .mu.m and 0.90 .mu.m,respectively, as measured by the manufacturer. The coefficient ofvariation (CV) of particle diameter .phi. is defined asCV=.DELTA..PHI.<.PHI.>.times. 100 .times. %, where .DELTA..phi. is thestandard deviation of .phi., and <.phi.> the mean of .phi.. The peakwidths measured at 11 mm from the injection point, correspond to CV's of2.5%, 1.2% and 0.9% for these three sizes, respectively (FIG. 21). CV'sare calculated according to: CV=d .PHI. d x .times. .DELTA. .times..times. x<.PHI.>.times. 100 .times. %, where x is the lateral positionof the band and .DELTA.x the measured standard deviation (half-width).Note that the measured peaks of 0.80 .mu.m and 0.90 .mu.m are as sharpas the size-variances of the microspheres themselves, and thus bandbroadening in our device is less than the known variance in particlesize.

Example 2

One advantage of the flexibility of microfabrication is that the arraycan be designed to have varying gap widths as a function of distance,thereby optimizing separation for complex mixtures. To demonstrate thispoint, a device was fabricated containing 9 sections, each of which hada different gap width, starting with 1.4 .mu.m and ending with 2.2 .mu.min increments of 0.1 .mu.m (FIG. 23 and FIG. 24). The varying gap widthswere designed to tune the critical diameter in 9 stages from .about.0.70.mu.m to .about.1.10 .mu.m, so that a given sized particle would switchfrom displacement mode to zigzag mode as the gap width increased.

A mixture of monodisperse (CV=1%) microspheres of 0.80 .mu.m, 0.90 .mu.mand 1.03 .mu.m was injected from the small-gap side of the array (FIG.23 and FIG. 24), and flown at .about.400 .mu.m/s using a drivingpressure of 30 kPa. Initially, all microspheres were larger than thecritical size, and migrated at the same angle with respect to thevertical flow (displacement mode). Soon, however, the 0.80 .mu.mmicrospheres (left) switched to the flow direction (vertical),presumably in zigzag mode. 0.90 .mu.m microspheres switched to verticalat the fourth section, and the 1.03 .mu.m microspheres made thetransition around the eighth section. The fluorescent intensity profilewas scanned at .about.14 mm from the injection point (FIG. 25), andshowed that the 0.80 .mu.m, 0.90 .mu.m and 1.03 .mu.m peaks had CV's of1.1%, 1.2% and 1.9%, respectively (see the scale bars, centered at themeans of the peaks). By comparison, the 1% CV attributable tononuniformity in the microsphere population is shown as the black scalebars underneath the peaks. Note that a fraction of the 1.03 .mu.mmicrospheres were separated out from the main peak and formed asub-band, most likely because of the non-homogeneity in the microspherepopulation. Again, the peaks are virtually resolved to themono-dispersity of the most uniform microspheres commercially available.The running time was .about.40 s.

The results of FIG. 21 and FIG. 25 suggest that the resolution of thedevice may exceed our ability to measure it. One could in principlediscover the limits of resolution for the approach by exploring theresolving power of the current array with a series of size classesvarying in approximately 0.01 .mu.m intervals around .about.1 .mu.m,each class with a CV of .about.0.1%.

Example 3

A device having identical array dimensions and loading structures as inExample 1 was constructed for separation of nucleic acids according tomolecular weight. The device was made of fused silica instead of siliconusing the same techniques as described in Example 1. Electric fields,instead of pressure-driven fluid flow, were used as the field. A mixtureof coliphage .lamda. and T2 dsDNA (48.5 kb and 164 kb respectively) at.about.2 .mu.g/ml and .about.1 .mu.g/ml was used as a test sample, andvisualized by fluorescent microscopy. DNA was stained with TOTO-1(Molecular Probes) at a ratio of 1 dye molecule per 10 base pairs. 0.1%POP-6, a performance-optimized linear polyacrylamide (Perkin-ElmerBiosystems), and 10 mM dithiothreitol (DTT) were added to the ½.times.Tris-Borate-EDTA buffer to suppress electro-osmotic flow andphoto-bleaching, respectively. The electric fields were applied usingelectrodes immersed in buffer reservoirs. The migration speeds of themolecules were controlled by the voltage applied to the reservoirs, andmeasured by observing the velocity of individual molecules.

The two species were separated with good resolution using an electricfield of .about.5 V/cm (FIG. 26).

Example 4

A device of the present intervention for concentrating DNA molecules ismade and schematically shown in FIG. 28. The device comprises an arrayand microfluidic channels for sample injection and extraction, which areidentical in dimensions with Example 1. The field used herein is anelectric field, applied using electrodes immersed in the buffer fluidreservoirs. The device is made of fused silica using techniquesdescribed in Example 1. Coliphage T2 DNA at .about.1 .mu.g/ml in½.times. Tris-Borate-EDTA buffer containing 0.1% POP-6, aperformance-optimized linear polyacrylamide (Perkin-Elmer Biosystems)and 10 mM dithiothreitol (DTT), was used as a test sample. The DNA wasloaded onto the device at the sample reservoir, and concentrated againsta boundary of the array to .about.120 .mu.g/ml (FIG. 29). The electricfield strength was .about.5 V/cm. The concentrated sample is removedfrom the array and collected in the sample-collection reservoir (FIG.28).

While the present invention is described with respect to particularexamples and preferred embodiments, it is understood that the presentinvention is not limited to these examples and embodiments. The presentinvention as claimed therefore includes variations from the particularexamples and preferred embodiments described herein, as will be apparentto one of skill in the art.

1. A microfluidic device for concentrating particles, having at least apredetermined critical size, the device comprising: a microfluidicchannel, an array of obstacles within said microfluidic channel, and aboundary, wherein the device employs a field that propels particlesthrough the microfluidic channel and said array of obstacles isasymmetric with respect to the average direction of the field, suchthat, when particles are introduced into the array, particles having asize less than a predetermined critical size are transported in a firstdirection, and particles having a size at least that of the criticalsize are transported in a second direction to the boundary, and whereinthe first and second directions are different, thereby concentrating theparticles having a size at least that of the critical size.
 2. Themicrofluidic device of claim 1, the microfluidic device furthercomprising a buffer reservoir at a beginning of said microfluidicchannel.
 3. The microfluidic device of claim 1, the microfluidic devicefurther comprising a sample reservoir at a beginning of saidmicrofluidic channel.
 4. The microfluidic device of claim 1, themicrofluidic device further comprising an unloading channel at an end ofsaid microfluidic channel.
 5. The microfluidic device of claim 1, themicrofluidic device further comprising a concentrated sample reservoirat an end of said microfluidic channel.
 6. The microfluidic device ofclaim 1, the microfluidic device further comprising: a buffer reservoirat a beginning of said microfluidic channel; a sample reservoir at abeginning of said microfluidic channel; an unloading channel at an endof said microfluidic channel; and a concentrated sample reservoir at anend of said microfluidic channel.
 7. The microfluidic device of claim 1,the microfluidic device further comprising a plurality of concentratedsample reservoirs at an end of said microfluidic channel, wherein eachsample reservoir receives a different size range of particles.
 8. Themicrofluidic device of claim 1, wherein said array of obstaclescomprises obstacles arranged in rows, and wherein at two adjacentobstacles in at least one row are separated by 8 μm center-to-center. 9.The microfluidic device of claim 1, wherein at least two laterallyadjacent obstacles in said array of obstacles are separated by between1.4 μm to 2.2 μm.
 10. The microfluidic device of claim 1, wherein atleast two laterally adjacent obstacles in said array of obstacles areseparated by 1.6 μm.
 11. The microfluidic device of claim 1, whereinsaid array of obstacles is tilted at an offset angle of 5.7° withrespect to the direction of the field.
 12. The microfluidic device ofclaim 1, wherein said array of obstacles comprises obstacles arranged inrows, and wherein each subsequent row of obstacles is shifted laterallywith respect to the previous row by between 0.0 to 0.5 times thedistance, center-to-center, of adjacent obstacles in a row.
 13. Themicrofluidic device of claim 1, wherein said array of obstaclescomprises obstacles arranged in rows, and wherein each subsequent row ofobstacles is shifted laterally with respect to the previous row byone-third the distance, center-to-center, of adjacent obstacles in arow.
 14. The microfluidic device of claim 1, wherein said array ofobstacles comprises rows of obstacles, and wherein at least one row ofobstacles comprises differently shaped obstacles than at least one otherrow of obstacles.
 15. The microfluidic device of claim 1, wherein saidarray of obstacles comprises a plurality of sub-arrays of obstacles, andwherein at least one sub-array of obstacles is different than at leastone other sub-array of obstacles.
 16. The microfluidic device of claim1, wherein said microfluidic channel is curved.
 17. The microfluidicdevice of claim 1, wherein the field comprises a plurality ofsub-fields, and wherein at least one sub-field has a different averagedirection of at least one other sub-field.
 18. The microfluidic deviceof claim 1, wherein the particles are bacteria, cells, organelles,viruses, nucleic acids, proteins, protein complexes, polymers, powders,latexes, emulsions, colloids, or biomolecules.
 19. The microfluidicdevice of claim 1, wherein said array of obstacles comprises an array ofcylindrical obstacles.
 20. The microfluidic device of claim 1, whereinsaid array of obstacles is fabricated from glass, fused silica, siliconerubber, silicon, ceramic, polymer, or plastic.
 21. A method comprising:introducing particles into a microfluidic channel comprising an array ofobstacles within said microfluidic channel, and a boundary, wherein thedevice employs a field that propels particles through said microfluidicchannel and said array of obstacles is asymmetric with respect to theaverage direction of the field, such that, when particles are introducedinto said array of obstacles, particles having a size less than apredetermined critical size are transported in a first direction, andparticles having a size at least that of the critical size aretransported in a second direction to the boundary, and wherein the firstand second directions are different, thereby concentrating or separatingthe particles having a size at least that of the critical size.
 22. Themethod of claim 21, wherein the method further comprises introducing abuffer into the microfluidic channel.
 23. The method of claim 21,wherein the method further comprises retrieving the concentrated orseparated particles having a size at least that of the critical size.24. The method of claim 21, wherein the method further comprisesretrieving the particles having a size less than the critical size. 25.The method of claim 21, wherein the method further comprises retrievingparticles from a plurality of concentrated sample reservoirs at an endof said microfluidic channel, wherein each sample reservoir receives adifferent size range of particles.
 26. The method of claim 21, whereinthe method further comprises providing the field such that the fieldcomprises a plurality of sub-fields, and wherein at least one sub-fieldhas a different average direction of at least one other sub-field.